[0001] The present invention relates to highly elastic and highly strong moulded materials
having excellent tensile strength and stretch resistance which are obtained from cellulose
produced by bacteria.
[0002] The moulded materials can be utilized not only as paper and as various other sheets
but as thread-like mouldings or as various solid mouldings.
[0003] As cellulose produced by bacteria, there is known a sheet-like cellulose produced
by Acetobacter xylinum ATCC 23769 for medical pads (see JP-A-120159/85).
[0004] On the other hand, various materials are known as conventional moulded materials.
Thus, cellulose, cellophane, celluloid, etc., obtained by dissolving cellulose derivatives
and then finishing, in addition to fibres-for forming thread-like mouldings, sheet-like
mouldings and various solid mouldings, are known. Furthermore, various synthetic high
molecular weight materials have also been developed and include those having particularly
improved dynamic strength due to the orientation of the molecular chains in a definite
direction.
[0005] The.dynamic strength of conventional cellulose and cellulose derivatives derived
from various plants is not very great. For example, the modulus of elasticity of celluloid
or cellophane in sheet form is approximately 2 to 3 GPa at the most.
[0006] Furthermore, synthetic high molecular weight materials obtained by orientating the
molecular chain in a definite direction-have, of necessity, a limited utility due
to poor modulus of elasticity in the other directions. This makes these materials
useless as strong materials, even though some of then have a modulus of elasticity
comparable to that of metals or inorganic materials in one direction. For this reason,
materials having no anisotropy in their molecular orientation but having excellent
strength as structural materials are desired. However, high molecular weight materials
in which the molecules are arranged at random have a poor modulus of elasticity. As
moulded materials having a high efficiency, synthetic high molecular weight materials
such as polyester films, aramide sheets, polyimide films and the like, are known but
the modulus of elasticity thereof is approximately 4 to 7 GPa.
[0007] As materials utilizing cellulose produced by bacteria, the foregoing materials are
known but their utility is limited to medical pads. Nothing is known as to the use
of these materials as high dynamic strength materials.
[0008] An object of the present invention is to provide highly elastic and highly strong
moulded materials which are excellent in tensile strength and resistance to stretching,
as compared to conventional moulded materials.
[0009] Another object of the present invention is to provide moulded materials having excellent
hydrophilic properties without problem as to toxicity, in addition to the high dynamic
strength.
[0010] A further object of the present invention is to provide materials having high dynamic
strength which are excellent for use in various fields due to the fact that they have
imparted thereto electrical conductivity, magnetic properties, highly insulating properties,
thermal conductivity, resistance to weather, resistance to chemicals, and so on.
[0011] The present inventors have made various investigations with an attempt to attain
these objects and found that cellulose comprising ribbon-like microfibrils produced
by microorganisms has an extremely large dynamic strength, such as tensile strength,
etc., and that the foregoing objects can be achieved by using, as moulded materials,
materials having incorporated therein this bacteria-produced cellulose.
[0012] Thus,, the present invention relates to moulded materials having a high dynamic strength
which comprise bacteria-produced cellulose comprising ribbon-like microfibrils.
[0013] The bacteria-produced cellulose comprises ribbon-like microfibrils which are preferably
approximately 100 to 500 Angstroms in width and approximately 10 to 200 Angstroms
in thickness.
[0014] The content of bacteria-produced cellulose of the material may be from 0.01 to 100%.
[0015] Preferably, the material additionally contains at least one material selected from
(a) a hydrophilic high molecular weight material, (b) a hydrophobic high molecular
weight material, (c) a metal, (d) an inorganic material, (e) a substa'nce which is
magnetic, (f) a substance which is electrically conductive, (g) a substance having
a high thermal conductivity, (h) a substance having a high resistance to weathering,
and (i) a substance having high resistance to chemicals.
[0016] The bacteria-produced cellulose is readily decomposed by cellulase to form glucose.
For example when cellulase (EC 3.2.1.4) (manufactured by Amano Pharmaceutical Co.,
Ltd.) was dissolved in a 0.1% (w/v) suspension of the cellulose in an amount of 0.5%
(w/v) and the mixture was reacted at 30
0C for 24 hours in a 0.1M acetate buffer, it.was observed that a part of the cellulose
decomposed. The supernatant was developed by paper chromatography, whereby cello-oligosaccharides
and glucose were detected. In addition, a small quantity of hexoses, other than glucose,
were detected.
[0017] Thus, the bacteria-containing cellulose of the present invention contains cellulose,
and it may contain heteropolysaccharides having cellulose as a main chain and β-1,3-,
β-1,2-, etc., glucans.
[0018] In the case of heteropolysaccharides, the constituent components, other than cellulose,
are hexose, pentose and organic acids, etc., such as mannose, fructose, galactose,
xylose, arabinose, ramnose, glucuronic acid, etc. These polysaccharides may be single
substances; alternatively, two or more polysaccharides may be combined via, for example,
a hydrogen bond, etc.
[0019] Any bacteria-produced cellulose is usable in the present invention.
[0020] Microorganisms that produce such cellulose are not particularly limited, and include
Acetobacter aceti subsp. xylinum ATCC 10821, Acetobacter pasteurianus, Acetobacter
rancens, Sarcina ventricuii, Bacterium xylinside and bacteria belonging to the genus
Pseudomonas, the genus Agrobacterium, etc.
[0021] The methods for culturing these microorganisms and accumulating bacteria-produced
cellulose are conventional methods for culturing bacteria. Thus, microorganisms are
inoculated on conventional nutrient media containing carbon sources, nitrogen sources,
inorganic salts and, if necessary, organic trace nutrients such as amino acids, vitamins,
etc. followed by settling or gentle aerial shaking. As carbon sources, glucose, sucrose,
maltose, starch hydrolysates, molasses, etc., may be used but ethanol, acetic acid,
citric acid, etc., may also be used singly or in combination with theabove-desribed
sugars. As nitrogen sources, organic or inorganic nitrogen sources such as ammonium
salts, e.g. ammonium sulphate, ammonium chloride, ammonium phosphate, etc., nitrates,
urea, peptone or the like may be used. Inorganic salts, phosphates, magnesium salts,
calcium salts, iron salts, manganese salts, etc., may be used. As organic trace nutrients,
amino acids, vitamins, fatty acids, nucleic acids, etc., may be used. Furthermore,
peptone, casamino acid, yeast extracts, soybean protein hydrolysates, etc., containing
these nutrients may be used. When using auxotrophs requiring amino acids, etc., for
growth, it may be necessary to use further nutrients.
[0022] The culture conditions may be conventional; for example, at a pH of 3 to 9 and at
a temperature of 20 to 40
oC, culture may be performed for 1 to 30 days, whereby the bacteria-produced cellulose
accumulates as a surface layer in a gel form.
[0023] The gel may be withdrawn and washed with water, if necessary. Depending upon the
intended use of the gel, the washing water may contain chemicals such as sterilizers,
pre-treating agents, etc.
[0024] After washing with water, the gel may be dried or kneaded with other materials, for
use.
[0025] It is preferred that the bacteria-produced cellulose be of structure in which the
microfibrils are intertwined, in order to enhance the dynamic strength such as tensile
strength, etc. For this reason, a method which for example, comprises pressing the
gel, withdrawn from the culture, from the orthogonal direction, squeezing most of
the free water off and then drying it, is effective. It is appropriate that the squeezing
pressure be approximately 1 to 10 kg/cm
2. By this press squeezing, the cellulose after drying is orientated along the press
squeezing direction. Furthermore, by stretching in one direction while applying pressure,
e.g. by performing a rolling operation, the cellulose after drying is orientated also
in the rolling direction, in addition to the press squeezing direction. Pressing apparatuses
can be appropriately chosen from commercially available machines.
[0026] On the other hand, it is also effective to macerate the bacteria-produced cellulose,
to increase the dynamic strength. Maceration may be carried out by using a mechanical
shearing force. The bacteria-produced cellulose can easily be macerated with, for
example, a rotary macerator, a mixer, etc. It is also effective to conduct the aforesaid
press squeezing after maceration.
[0027] The moulded material having a high dynamic strength of the present invention can
be in the form of various shapes such as sheet-like shapes, yarn-like shapes, cloth-like
shapes, solid-like shapes, etc.
[0028] In the case of moulding into a sheet-like form, the bacteria-produced cellulose is,
if desired, macerated and then formed into a layer, which is squeezed under pressure,
if desired, and then dried. By press squeezing, a'planar-orientated sheet is obtained;
by further rolling, a sheet not only planar- orientated but also uniaxially orientated
can be obtained.
[0029] It is desired that the drying of the sheet, macerated and/or press squeezed, be carried
out after fixing it to a suitable support. By fixing it on a support, the degree of
planar-orientation is further enhanced and a sheet having a large dynamic strength
can be obtained. As supports, plates, e.g. glass plates, metal plates, etc., having,
for example, a net structure, can be used. Any drying temperature can be used as long
as the temperature is within a range where the cellulose is not decomposed. In addition
to heat drying, freeze drying can also be used.
[0030] The thus obtained sheet has a structure in which the microfibrils are intertwined
at random. According to an X-ray diffraction pattern, the press-squeezed sheet is
planarily orientated; the additionally rolled sheet is planarily orientated and, at
the same time, uniaxially orientated- The modulus of elasticity of the sheet is generally
about 10 to 20 GPa.
[0031] The thickness of the sheet depends upon its intended use, but is generally about
1 to 500 microns.
[0032] The sheet may contain various additives. For example, by incorporating solutions
(aqueous or nonaqueous), emulsions, dispersions, powders, melts, etc. of various high
molecular weight materials, one or more of strength, weatherproofness, chemical resistance,
water resistance, water repellency, antistatic properties, etc., can be imparted to
the sheet, depending upon the properties of the additives. By incorporating metals
such as aluminium, copper, iron, zinc, etc., or carbon in a powdery form or fibre
form, electroconductivity and thermal conductivity can be increased. Further by incorporating
inorganic materials such as titanium oxide, iron oxides, calcium carbonate, kaolin,
bentonite, zeolite, mica, alumina, etc., the heat resistance, insulating properties,
etc., can be improved or smoothness can be imparted to the surface, depending upon
kind thereof. By incorporating lower molecular weight organic materials or adhesives,
the strength can be further increased. The sheet may be coloured with colouring agents
such as phthalocyanine, azo compounds, indigos, safflowers, etc. For colouration,
various paints, dyes and pigments can be used in addition thereto. By incorporating
medicines or sterilizers, the sheet can also be utilized.as a medical sheet.
[0033] These kneadins and additives are incorporated in an appropriate amount not exceeding
97% by imparting the desired physical properties. The time of the incorporation is
not limited, and they may be incorporated in the bacteria-produced cellulose gel or
a macerated product thereof; alternatively, they may be incorporated after press squeezing
or after drying. Furthermore, they may be incorporated in the media or culture on
in some occasions. The method of incorporation may be by impregnation, in addition
to mixing.
[0034] On such a sheet can also be laminated a layer of other material. The laminate can
be appropriately chosen depending upon the intended purpose of the sheet. The laminate
can also be chosed from the aforesaid kneadings or additives; for example, various
high molecular weight materials can be coated onto the sheet to impart waterproofness
to the sheet.
[0035] In the case of paper, the bacteria-produced cellulose can be macerated, then subjected
to paper making and then drying, whereby paper having an excellent tensile strength,
resistance to expansion, etc., and at the same time having a high elasticity and a
high strength and which is chemically stable and excellent in water absorbance and
air permeability can be obtained. In this case, ordinary additives, treating agents,
etc., used for paper making can be utilized and kneadings and additives can also be
appropriately chosen from the aforesaid substances and incorporated into the paper.
[0036] In recent years, the demand for electrically insulating paper, heat resistant paper,
fire retarding paper, etc., has increased and synthetic paper, inorganic paper, etc.,
containing non-cellulose fibres have been prepared. When one attempts to prepare such
paper by a wet method, it is necessary to perform paper making by the use of cellulose
pulp, because non-cellulose fibres fail to form hydrogen bonds except where pulp fibres
such as fibres of polyethylene, polypropylene, polyacrylonitrile, aromatic polyamide,
etc., are used. In this case, it is necessary that the amount of pulp be minimized
as much as possible to enhance the insulating, heat resistant and fire retarding properties.
In the case of mixing conventional wood pulp with paper, however, the amount added
reaches 20 to 50% and the intended effect cannot be sufficiently achieved. On the
contrary, the amount of cellulose can be greatly reduced by the use of the bacteria-produced
cellulose instead of wood pulp and paper having excellent insulating, heat resistance
and fire retarding properties can be obtained. Accordingly, the moulded material having
a high dynamic strength of the present invention is also effective in this respect.
[0037] Furthermore, it is said that photo-crosslinking polyvinyl alcholol has a good affinity
to living things as compared to conventional photo-crosslinking resins and it is thought
that the utility of immobilizing agents for enzymes, microorganisms, etc. would be
further improved. A photoresist used for making an original printing plate can be
prepared by coating a resin on a base plate, by projecting a design, etc., to be printed
thereon to cause photo-crosslinking and hardening of the resin, and by washing off
unhardened resin. The photo-crosslinkable polyvinyl alcohol, which is water-soluble,
has advantages in that it is inexpensive and the washing thereof is easy as compared
to conventional oil-soluble photoresists, and is expected to be useful as a photoresist.
In this case, however, there is a problem in that photo-crosslinkable polyvinyl alcohol
swells in water and the cross-linked structure is destroyed. However, this swelling
can be prevented by incorporating bacteria-produced cellulose into it.
[0038] In the case of yarn, for example, the bacteria-produced cellulose gel or macerated
product thereof may be washed, dried and then dissolved in, for example, a dimethylacetamide/lithium
chloride solvent. The solution is spun using a coagulating solution such as water,
alcohols, ketones, dioxane, tetrahydrofuran, etc., in which cellulose is insoluble
and the solvent for the cellulose is miscible.
[0039] In the case of cloth, this yarn may be woven in a conventional manner.
[0040] In the case of a solid structure, various plastics materials may be kneaded with
or laminated on the bacteria-produced cellulose to form the desired moulding. This
moulding is substitutable for, example, various Cibre-reinforced plastics products
or carbon fibre products.
[0041] The bacteria-produced cellulose comprises ribbon-like microfibrils and has large
dynamic strengths such as tensile strength, expansion resistance, elasticity, etc.
The dynamic strength is enhanced by the microfibrils being intertwin"ed. By imparting
orientation thereto, the strength in such a direction is further increased. The bacteria-produced
cellulose has the property that is readily orientated by pressing.
[0042] The moulded material'having a high dynamic strength of the present invention is excellent
in tensile strength, expansion resistance, elasticity, etc. In particular, the sheet
obtained after press squeezing and drying has an extremely high modulus of elasticity;
in the case of the articles of the Examples hereinbelow, the modulus of elasticity
was more than twice that of the sheet of polymetaphenyleneisophthalamide having the
highest modulus of elasticity among secondary materials heretofore known.
[0043] Accordingly, the material can be used as reinforcing material for composite plastics
materials requiring a high strength, for example, as a body material for ships, aircrafts,
automobiles, etc., as a printed circuit base, etc., as a high quality paper such as
recording paper, etc., or as diaphragm, etc., for percussion instruments.
[0044] The invention will now be illustrated by the following Examples, in which reference
is made to Figures 1 to 4 of the drawings, which Figures are explained in the relevant
Examples.
[0045] The bacteria-produced cellulose used in the Examples was derived from Acetobacter
aceti subsp. x
ylinum ATCC 10821 (which is available to the public, from the American Type Culture
Collection).
[0046] The following abbreviations are used in the Examples:
N.U.SP : soft wood unbleached sulphite pulp
CSF : Canadian Standard Freeness (TAPPI Bulletin T 227 M-58, May 1948)
L.B.KP : hard wood pulp bleached kraft paper.
Example 1
[0047] In a 200 ml Erlenmeyer flask, there was charged 50 ml of a medium consisting of 5
g/dl of sucrose, 0.5 g/dl of yeast extract (Difco), 0.3 g/dl of KH
2PO
4 and 0.05 g/dl of MgS0
4.7H
20, and having a pH of 5.0. The medium was sterilized with steam at 120
oC for 20 minutes. The sterilized medium was innoculated with a platinum loop of Acetobacter
aceti subsp. xylinum ATCC 10821, grown at 30°C for 30 days in a test tube slant agar
medium (pH 6.0) consisting of 0.5 g/dl of yeast extract, 0.3 g/dl of peptone and 2.5
g/dl of mannitol (pH 6.0), and the medium was cultured at 30
oC. Thirty days thereafter, a gel-like membrane containing white bacteria-produced
cellulose polysaccharides had formed as a surface layer on the culture solution.
[0048] The thus obtained gel-like membrane was washed with water and spread in a layer having
a thickness of about 1 cm. By pressing the membrane under a pressure of about 10 kg/cm
2 using a test pressing machine (made by Tester Industry Co., Ltd.), water was squeezed
out. The layer was attached to a glass plate and dried at 105
0C for 2 hours to obtain a sheet having a thickness of about 10 microns.
[0049] The X-ray diffraction pattern of the sheet thus obtained is shown in Figure 1. This
Figure shows the diffraction pattern obtained by having its rotation axis parallel
to the sheet plane and photographing with an X-ray incident upon the direction orthogonal
to the rotation axis. As shown in the Figure, plane 101, plane 101, and plane 002
are all orientated, and this sheet undergoes planar orientation to an extremely high
degree.
[0050] The modulus of elasticity of this sheet, of known cellulosic sheets and of various
high molecular weight secondary materials were measured using a tensile tester. The
results are given in Table 1.

Example 2
[0051] Gel-like bacteria-produced cellulose, as used in Example 1, was squeezed under pressure
while rolling it in one direction using a roll pressing machine (made by Yoshida Kogyo
K.K.). In the same manner as Example 1, the squeezed cellulose was attached to a glass
plate and dried at 105°C for 2 hours to obtain a sheet.
[0052] The X-ray diffraction pattern of the sheet thus obtained is shown in Figure 2. This
Figure shows the diffraction pattern obtained by fixing the sheet and photographing
it with an X-ray incident upon the direction perpendicular to the film surface. In
the Figure, the arrow indicates the direction of rolling. As shown in the Figure,
orientation is found in each of plane 101, plane 101 and plane 002, and uniaxial orientation
is obviously clear. Furthermore, with respect to planar orientation, orientation almost
similar to that of Figure 1 is clear.
[0053] The modulus of elasticity of this sheet was measured in a manner similar to Example
1, and was found to be 20 GPa in the rolling direction.
Example 3
[0054] Bacteria-produced cellulose was incorporated in novolid fibres (Kainol Fiber KF 0203,
14 microns in diameter and 3 mm in length, manufacutred by Gun-Ei Chemical Industry
Co., Ltd.) and a sheet having a weight of 60 g/m
2 was produced therefrom by paper making according to the TAPPI method (TAPPI standard
T 205 m-58).
[0055] Furthermore, for the purpose of comparison, papier mache was made from ordinary wood
pulp (N.U.SP), beaten to a high degree (CSF 245 ml), and Kainol Fiber.
[0056] The breaking length of each of these sheets was measured using an automatic recording
tensile tester. The results are given in Table 2.

[0057] By the use of bacteria-produced cellulose, paper making is possible and the strength
of the paper is increased, by the use of the bacteria-produced cellulose in a small
amount. In the case of using conventional pulp, paper making was impossible in the
case of the use of less than 10 parts.
Example 4
[0058] Using various inorganic fibres and bacteria-produced cellulose, papier mache was
prepared, and the breaking length was measured. The results are given in Table 3.

[0059] In each case, paper making was possible in the case of the use of 5 to 10% of the
bacteria-produced cellulose.
Example 5
[0060] Gel-like bacteria-produced cellulose was pressed and dried to obtain a sheet. The
Young's modulus (E) of the sheet, measured by the oscillation lead method, was 13.6
GPa.
[0061] This value is 5 to 10 times the Young's modulus of conventional paper prepared from
wood pulp alone.
Example 6
[0062] Gel-like bacteria-produced cellulose was macerated with a homogenizer, and the macerated
material was made into paper according to the TAPPI method. The Young's modulus (E)
of the sheet, measured by the oscillation lead method, was 7.4 GPa.
[0063] Furthermore, for the purpose of improving the freeness and yield of fine particles,
5% (ratio of solids contents) of polyamide epichlorohydrin resin (Kaimen 557H, manufactured
by Dick Hercules Co., Ltd.) was incorporated therein, and paper was made therefrom.
The Young's modules (E) of this paper was 8.1 GPa.
[0064] In each case, paper having a high strength was obtained.
Example 7
[0065] To bacteria-produced cellulose, there was added wood pulp (N.U.KP) (CSF, 540 ml).
Furthermore, 5% (ratio of solids contents) of aluminium sulphate_was added to the
mixture, and paper was made from the mixture. The properties of the paper thus obtained
were as given in Table 4.

[0066] By the addition of the bacteria-containing cellulose, the strength of the paper was
improved.
Example 8
[0067] After macerating gel-like bacteria-produced cellulose, obtained by definite culture,
with a standard pulp macerator, the macerated material was filtered using a 125 mesh
sieve to obtain macerated paste having a solids content of about 8.8%. The paste was
used in the following experiments.
[0068] Photo-crosslinkable polyvinyl alchol, (PVA)-SbQ (GH-17SbQ, 10.5 wt%, 1.2 mol%, manufactured
by Toyo Gosei Kyogyo K.K.), the macerated paste described above and water were mixed
in a dark room in the ratios given in Table 5.

[0069] Each mixture given in the Table above was spread over an acrylic plate in a thickness
of about 0.7 mm, using a glass rod. After air drying overnight, each mixture was further
air dried at 40
0C for 30 minutes. These operations were performed in a dark room. Each mixture was
exposed to sunlight for 30 minutes to cause crosslinking and to obtain a sheet.
[0070] The sheet was cut into a ribbon-like shape of 3 x 1 cm and put into water for 2 hours
to measure the extent of swelling. The results are given in Table 6.

[0071] By the use of the bacteria-produced cellulose, swelling could be prevented.
[0072] Next, tensile tests were performed on the sheets. The results are given in Table
7.

[0073] By using the bacteria-produced cellulose, the modulus of elasticity could be improved
to 1.3 to 1.6 times.
Example 9
[0074] Each of the mixtures given in Example 8 was sandwiched between two glass plates using
as a spacer a slide glass having a thickness of 1 mm. Each mixture was then exposed
to sunlight for 30 minutes to cause crosslinking and a gel similar to konjak (an edible
gel made from plant material) in a wet state was obtained. The gel was put into distilled
water and a swelling test was performed in a manner similar to Example 8. The results
are given in Table 8.

[0075] *Samples 3 and 4 were destroyed by swelling.
[0076] By using the bacteria-produced cellulose, swelling and destruction of the gel were
prevented.
Example 10
[0077] To 3.5 parts of dry bacteria-produced cellulose, there were added 100 parts of dimethylacetamide.
The mixture was refluxed with stirring for 60 minutes. Next, the mixture was cooled
to 100
oC. After slowly adding 10 parts of lithium chloride, the mixture was stirred at room-temperature
overnight to dissolve the bacteria-produced cellulose. The resulting spinning dope
was spun in a tetrahydrofuran coagulating solution, at a spinning draft of 1.5 and
at a bath length of 80 cm. The yarns from the spinning bath were stretched by 50%
in water and at 50°C and then dried. The properties of the fibres thus obtained are
given in Table 9. For the purpose of comparison, wood pulp (L.B. KP) was spun in a
similar manner.

Example 11
[0078] To a mixture of copper powder (manufactured by Fukuda Kinzokuhakufun Kogyo K.K-,
10 microns in diameter) and wood pulp (N.U. KP) (CSF, 540 ml), macerated bacteria-produced
cellulose was added, and the mixture was subjected to paper making by the TAPPI method.
Furthermore, for the purpose of comparison, papier mache was also prepared from copper
powder and wood pulp. The physical properties thereof were measured using an automatic
recording tensile tester. The results are given in Table 10.

[0079] By the use of the bacteria-produced cellulose, the copper powder did not egress and
the strength of the paper was greatly improved. In the case of the conventional pulp,
more than 60% of the copper powder egressed.
Example 12
[0080] To wood pulp (N.U.KP) (CSF, 540 ml), there was added macerated bacteria-produced
(BC), and the mixture was subjected to paper making by the TAPPI method. The resulting
paper was impregnated with a phenolic resin and air dried. By hot press finishing,
a phenolic laminated plate was prepared. Furthermore, for the purpose of comparison,
a phenolic laminated plate comprising wood pulp alone was also prepared in a similar
manner. These phenolic laminated plates were moulded into Dumbell Moldel No. 1 (JIS
K-7113) and the physical properties thereof were measured using an automatic recording
tensile tester. The results are given in Table 11.

[0081] By the use of the bacteria-produced cellulose, the strength of the phenolic laminated
plate was greatly improved.
Example 13
[0082] Gel-like bacteria-produced cellulose as used in Example 1 was subjected to hot pressing
(using a machine made by Yoshida Kogyo K.K.) at 150°C under a pressure of 5 kg/cm
2 for 5 minutes to obtain a sheet. A polyethylene-imine-treated polyethylene film was
laminated onto the thus obtained sheet at 320°C to prepare a laminated film. The physical
properties of the film were measured using an automatic recording tensile tester.
The laminated film had a greatly improved modulus of elasticity, i.e. 16.2 GPa, as
compared to ordinarly cellophane-polyethylene laminated film, which had a modulus
of elasticity of 1.7 GPa.
Example 14
[0083] To silicon nitride and silicon carbide (manufactured by Tateho Chemical Co., Ltd.,
10 microns in length) there was added macerated bacteria-produced cellulose (BC),
and the mixture was subjected to paper making by the TAPPI method. Furthermore, for
the purpose of comparison, papier mache made of wood pulp (N.U.KP) and microfibril
cellulose (MFC, manufactured by Daicel Chemical Co., Ltd.) was also treated in a similar
manner. The physical properties of the sheets obtained were measured using an automatic
recording tensile tester. The results are given in Table 12.

[0084] By the use of the bacteria-produced cellulose, no egression of silicon nitride and
silicon carbide occurred but the modulus of elasticity was greatly improved. In the
case of ordinary pulp, 60% or more of silicon nitride and silicon carbide egressed.
In the case of MFC, most of the silicon nitride and silicon carbide egressed.
Example 15
[0085] In a 500 ml Sakaguchi flask, there was charged a 50 ml aliquot of a liquid medium
consisting of 2 g/dl of fumaric acid, 0.2 g/dl of dihydrogen potassium phosphate,
1 mg/dl of magnesium sulphate tetrahydrate, 1 mg/dl of magnesium sulphate heptahydrate,
0.05 g/dl of calcium chloride, 1.0 g/dl of yeast extract (Difco) and 1.0 g/dl of peptone
(Difco), which medium was adjusted to a pH of 7.0 using ammonia. One platinum loop
of E. coli ATCC 11775 was inoculated on the medium followed by culturing at 30°C for
24 hours. Then, the bacteria were recovered by centrifugation in a conventional manner.
After washing twice with physiological saline, the bacteria were suspended in the
same amount of physiological saline as their wet weight.
[0086] By the following method, an attempt was made to immobilize.the bacteria on a carrier
prepared from gelatin and cellulose. As the method for immobilization, the method
using transglutaminase, as described in JP-A-66886/84, was used. Gelatin (supplied
by Miyagi Kagaku) and the macerated cellulos ic substance were mixed in ratios given
in Table 13. To the mixture, the bacteria were added in an amount of 3.5% and the
mixture was gelled in the manner described in JP-A-66886/84. Thus, 0.1 unit was added
per 1 mg of transglutaminase. The mixture was allowed to stand at 25°C for 1 hour
to cause gellation. The gel was cut into cubes 5 mm square and added to a reaction
mixture to examine the aspartase activity. The reaction mixture contained 20 g/dl
of fumaric acid and 1mm of MgS0
4.7H
20, and the pH was adjusted to 8.5 using ammonia. Bacteria-immobilized gelatin get
was added to the reaction mixture in a bacteria concentration of 0.5% based on the
total amount of the reaction mixture. A reaction was carried out for 1 hour. Every
5 minutes the concentration of aspartic acid was quantitatively assayed by ninhydrin
colorimetry to determine the initial reaction rate. The count of the bacteria egressed
into the reaction mixture was determined by a colony count after 30 minutes from the
initiation of the reaction. The results are given in Table 13.

[0087] The enzyme activity was determined from the initial reaction rate. The enzyme activity
was expressed by a relative value, taking as 100 the case of the addition of no cellulosic
substance. Further the reaction was repeated 10 times. The results are given in Table
14.

[0088] By the incorporating the cellulosic substance, the egression of the bacteria was
prevented so that it became possible to maintain the activity after repeated use.
[0089] Furthermore, the breaking strength of gel supplemented with gelatin and reinforcing
materials similar to those given in Table 13 was measured. The measurement was performed
by expressing as a breaking strength the maxium load obtained by causing gellation
of the aforesaid gelatin solution in a cylindrical plastic container having a diameter
of 22 cm, then setting it in a rheometer (Fudo Kogyo K.K., NRM 2002 J) and immersing
an adapter of 5 mm diameter directly into the gel. The results are given in Table
15.

[0090] With the cellulosic material, a reinforcing effect superior to that of a conventional
reinforcement was noted.
Example 16
[0091] Immobilization to alginic acid gel was conducted by mixing sodium alginate with the
cellulosic substance according to Table 16. Thereafter the mixture was added dropwise
to a 0.1M CaC1
2 solution to obtain a bead-like gel.
[0092] An aspartase reaction was carried out under the same reaction conditions as used
in Example 15. The count of bacteria egressed was determined by the number of colonies
after 30 minutes, without exchanging the reaction mixture. The results are given in
Table 16.

[0093] The reaction solution described above was replenished every 15 minutes, and the reaction
of the immobilized carrier was carried out 4 times in total. The enzyme activity was
determined from its initial rate by measuring the concentration of aspartic acid every
3 minutes. The results are given in Table 17. The aspartase activity is shown by a
relative value, the value first obtained when no cellulosic substance was added being
100. The results are given in Table 17.

[0094] As a result of using the cellulosic substance as a reinforcement, the egression of
the bacteria was prevented so that it became possible to repeatedly use the immobilized
carrier, having the high strength of wood pulp as compared to the conventional immobilized
carrier.
Example 17
[0095] In the following manner, invertase was immobilized onto a photo-crosslinkale resin.
A mixture of 1 part of invertase and 2 parts of phosphate buffer (pH 6.0) was mixed
with 20 parts of a photo-crosslinkable resin solution (pH 6.0). The mixture was spread
over a glass plate. After air drying for 1 day, irradiation with light was performed
for 1 hour to cause crosslinking and hardening. The cellulosic substanoe was incorporated
in a final concentration of 5%.
[0096] The immobilized membrane was cut into a size of 5 x 5 mm and reacted in 50 ml of
a solution of 4 g of sucrose at 40
0C for 24 hours with stirring to examine the decomposition rate of sucrose. The reaction
was repeated 10 times. Furthermore, the breaking stress and modulus of elasticity
were also examined. The results are given in Table 18.

[0097] By incorporation the cellulosic substance into the membrane, the physical properties
of the membrane such as the breaking shear, modulus of elasticity, etc., representing
the strength of the immobilized enzyme membrane, were greatly improved. For this reason,
the immobilization of the enzyme was easy and the time required for the immoblization
in the case of the use of the cellulosic substance was shortened to 75% as compared
to the case in which no cellulosic substance was used.
Example 18
[0098] In a 200 ml Erlenmeyer flask, there was charged 50 ml of a medium (pH 5.0) consisting
of 5 g/dl of sucrose, 0.5 g/dl of yeast extract, 0.5 g/dl of ammonium sulphate, 0.3
g/dl of hydrogen potassium phosphate (KH
2P0
4) and 0.05 g/dl of magnesium sulphate (MgS0
4.7H
20). The medium was sterilized with steam at 120
0C for 20 minutes to form a culture solution.
[0099] Then, the culture solution was inoculated with one platinum loop of Acetobacter aceti
subsp. xylinum (ATCC 10821), grown at 30°C for 3 days in a test tube slant agar medium
(pH 6.0) consisting of 0.5 g/dl of yeast extract, 0.3 g/dl of peptone and 2.5 g/dl
of mannitol, and the culture solution was cultured at 30
0C for 30 days under the conditions described above whereby a gel-like membrane containing
white bacteria-produced cellulose polysaccharides was formed in the upper layer of
the culture solution. This gel-like membrane of cellulosic polysaccharides was washed
with water to obtain bacteria-produced cellulose.
[0100] The thus obtained bacteria-produced cellulose was inserted between metal plates and
press dried at 130
oC to obtain a sheet of bacteria-produced cellulose.
[0101] The physical properties of the bacteria-produced cellulose sheet were measured by
the oscillation lead method. The results given in Table were obtained.

[0102] Then, a paper honeycomb was prepared using the sheet of bacteria-produced cellulose
as a skin agent. A core having a cell size of 3 mm, which had been immersed in 6%
phenol and which had a thickness of 2.8 mm, was used.
[0103] A flat speaker, 60 mm square, was prepared using the paper honeycomb as a diaphragm.
This is designated Sample 1.
[0104] For the purpose of comparison, a paper honeycomb was prepared using the same core
as Sample 1 and kraft paper as a skin agent. Using the paper honeycomb as a diaphragm,
a flat speaker was prepared in a manner similar to Sample 1, which speaker is designated
Comparative Sample 1. The physical properties of the kraft paper used herein are shown
in Table 20.

[0105] The frequency characteristics of Sample 1 and Comparative Sample 1 were measured.
The results are shown in Figure 3.
[0106] From Figure 3, it is noted that the high band reproducing threshold frequency is
shifted to a high frequency band and, at the same time, this peak becomes small in
the case of Sample 1, using the bacteria-produced cellulose sheet as the skin agent,
and thus the reproducing frequency band is greatly broadened.
Example 19
[0107] The bacteria-produced cellulose prepared in the foregoing Example was macerated in
a macerator. After adding 4% (ratio of solids contents) of rosin size and 4% (ratio
of solids contents) of aluminium sulphate as sizing agents, cone paper was prepared
by a conventional paper making process. The physical properties of the cone paper
were measured by the oscillation lead method. The results given in Table 21 were obtained.

[0108] Then, a full range speaker unit having a diameter of 12 cm was prepared using this
cone paper. This is designated Sample 2.
[0109] For the purpose of comparison, a full range speaker unit was prepared in a manner
similar to Sample 2, using cone paper prepared from ordinary kraft pulp, which unit
is designated Comparative Sample 2. The physical properties of the cone paper are
given in Table 22.

[0110] The frequency characteristics of Sample 2 and Comparative Sample 2 were measured.
The results are shown in Figure 4.
[0111] From Figure 4, it is noted that, in the case of Sample 2 prepared from cone paper
containing the bacteria-produced cellulose, the high band reproducing threshold frequency
was shifted to a high frequency and and the reproducing frequency band was broadened.
Example 20
[0112] A part of the wood pulp used for making ordinary cone paper was replaced (in amounts
of 5% and 15%) by the bacteria-produced cellulose obtained in Example 18, as shown
in Table 23, and paper making by the TAPPI method was carried out. Thus, cone paper
(Samples 3 and 4) was obtained. As the wood pulp, N.U.SP (freeness, 570 ml, Csf) was
used, and the bacteria-produced cellulose used was macerated bacteria-produced cellulose.
Furthermore, cone paper obtained by paper making using wood pulp alone was made, and
is designated Comparative Sample 3.
[0113] The physical properties of these samples were measured by the oscillation lead method.
The results are given in Table 23.

[0114] From Table 23, it is noted that by using the bacteria-produced cellulose, an improvement
in strength is obtained while internal loss is maintained.
[0115] As is apparant from the present Example and from Example 18 and 19, in the acoustic
diaphragm of the present invention, the bacteria-produced cellulose is used at least
as part of the cellulose fibres thereof, and a paper diaphragm having an extremely
high strength and a broadened reproducing frequency band is obtained.
[0116] Furthermore, in the case of the diaphragm of the present invention, there is no danger
of problems such as a reduction of internal loss, a sense of incompatibility in sound
quality, etc., because it is unnecessary to use non-cellulosic materials (which are
unable to form hydrogen bonds).